The aim of this doctoral thesis was to discover and synthesise molecules endowed with activity against TB and MDR-TB and to develop novel sustainable and faster synthetic methodologies for the synthesis of heterocycles for the production of drug-like compounds. To address these objectives, in the first section of this work was described the discovery of two novel classes of antitubercular agents, while in the second section, the development of two procedurally simple and fast methodologies for the synthesis of pyrroles were described. Within the first part of this work, two distinct strategies have been applied respectively to the research of two novel classes of antitubercular agents. The first strategy adopted was the molecular simplification approach on the structure of the third line drug thioridazine. Following the modification on the TZ scaffold described in paragraph 2.2, two series of derivatives were synthesised and assessed against a panel of Mycobacteria including clinical isolates. The analysis of the activity data of the set of thioridazine derivatives synthesised allowed the identification of the SAR summarized in Figure 31.
Figure 31. Diagrammatic summary of structure activity relationship of 1 derivatives.
The screening of TZ derivatives led to the identification of compound 16e: a thioridazine derivative obtained by the demethylation of the piperidine ring in addition to the replacement of the phenothiazine core with an indole heterocycle (Figure 32). 16e showed an activity profile better than that of TZ and a cytotoxicity about 15-fold lower toward both the cells lines MRC-5 and J774A.1. Despite the molecular target of this new class of molecules being still elusive, and the construction of a pharmacophoric model is still challenging to be
synthesis of putative 16e derivatives have the potential to lead to the identification of a novel class of potent antitubercular compounds and to increase the understanding on the mechanism of action of this new class of molecules.
Figure 32. Thioridazine derivative 16e and its activity profile.
In the second part of the first section of this work, a novel class of molecules, active against Mtb and MDR-TB, has been designed by the application of a molecular hybridization approach on the structures of the antitubercular agents BM212 (I) and SQ109 (II). The molecules synthesised have been evaluated for their activity against Mtb and MDR-TB clinical isolates leading to the identification of exhaustive SAR, which suggested the key features paramount for the antitubercular activity of this new class of pyrroles (Figure 33).
Figure 33. Diagrammatic summary of structure activity relationship of the hybrid
Five compounds showed MIC values on Mtb H37Rv at ≤1.0 μg/mL, and two of them (26b and 26c) proved to be highly active also against MDR-TB strains (Figure 34).
Figure 34. Superimposition of BM212 and SQ109 led to the discovery of hybrid
derivatives 26b and 26c.
Among the compounds which had the better antitubercular profile, 26c showed the best drug profile proving to be superior to BM212 in terms of activity, cytotoxicity, and potency toward MDR-TB clinical isolates, thus turning out to be an excellent lead candidate for preclinical trials. Moreover, compounds 24c, 24h, 24i, and 24l, bearing a bulky alkyl substituent on the piperazine ring, showed potent EPI activity, comparable to that of verapamil, turning out to be promising multi-drug resistance reversal agents.
Furthermore, the second section of this work was dedicated to the research of novel methodologies toward the synthesis of relevant building blocks for the production of drug- like compounds, in detail pyrroles . Therefore, two novel cascade processes involving the metathesis reaction for the production of functionalised pyrroles have been described. The first methodology allowed the synthesis of 1,2,3-substituted pyrroles through a one-pot tandem enyne cross metathesis (CM)-cyclization reaction starting from appropriate propargylamines and the inexpensive ethyl vinyl ether. The scope of the reaction was demonstrated by the synthesis with medium-good yields of a large variety of pyrroles bearing aryl, hetero-aryl and alkyl substituents (Table 32).
Table 32. Synthesis of 1,2,3-substituted pyrroles from propargylamines vis one pot enyne
CM-cyclization reaction.
The reaction is rapid, procedurally simple and represents a facile entry to the synthetically challenging 4,5-unsubstituted pyrroles. All the pyrroles obtained with this methodology have a methyl group at C3 deriving from the diene intermediate of the enyne metathesis reaction. Attempts to decorate directly the methyl group were unsuccessful due to the presence of the reactive CH at positions C4 and C5. However, the value of the methodology is corroborated by the conversion of pyrroles into 3-pyrrolines and the consequent derivatization of the methyl substituent in C3 (Scheme 20). Moreover, the reaction was used for the synthesis of compound 47 that is an important building block for the synthesis of an alkaloid from the poison gland of ants Leptothoracini 48 (Scheme 21). This application highlights the versatility and usefulness of this reaction toward the production of important pharmaceuticals of interest.
The second part of chapter 3 was dedicated to the discovery and exploration of a novel chemo-enzymatic process for the synthesis of pyrroles. Firstly, the precedent undisclosed aromatization property of monoamine oxidase (MAO-N & 6-HDNO) biocatalysts has been
unveiled for the first time within this work, converting a library of 3-pyrrolines in the corresponding pyrroles through whole cell MAO catalysed oxidation-aromatization reaction. This biotransformation represents the first application of MAO biocatalysts as aromatizing agents and a series of pyrroles bearing alkyl and aryl substituents were synthesised with high conversion factors and good yield.
Table 33. MAO-D5 aromatization of 3-pyrroline in pyrroles.
From an analysis of the biotransformation conversion factors is highlighted that the success of the oxidation of 3-pyrrolines in pyrroles by MAO biocatalyst heavily depends on steric factors and on electronic effects that the pyrroline ring substituents could have on the nitrogen. As a general trend, N-alkyl-substituted pyrrolines afford the corresponding pyrroles with higher conversion factors than the N-aryl pyrrolines. Furthermore, N-aryl pyrrolines bearing electron withdrawing groups on the aromatic ring are not processed by
MAO biocatalyst. This is mainly due to the reduced nucleophilicity of the pyrroline nitrogen of these substrates, as described in paragraph 3.3.1.3. Moreover, in the same paragraph it is described that steric factors prevent N-aryl pyrrolines decorated with a phenyl group on C2 or C3 to be converted in the corresponding pyrroles. Despite these limitations, the discovery of the aromatizing property of MAO biocatalysts make them a valid and sustainable alternative to the chemical or metal agents used for the oxidation of C-N bonds for the synthesis of pyrroles. Moreover, further directed evolution of this class of enzyme could lead to the discovery of a monoamine oxidase with a larger substrate scope expanding even more the application field for such biocatalysts. Additionally, in the last part of this work is described the first attempt to combine efficiently MAO oxidation reaction in the same reaction medium with RCM reaction in order to produce pyrroles by convenient starting materials such as opportunely substituted N,N-diallylamines and anilines (Table 34).
Table 34. Novel one-pot chemoenzymatic methodology toward the synthesis of pyrroles.
The main charm of this technique is the successful combination of chemo- and enzymatic catalysis in a concurrent fashion. Mimicking the compartmentalization of cellular processes
by appropriate choice of solvents and reaction conditions, it is possible to overcome the compatibility issues of the catalysts and the difference on the reaction conditions in which these generally operate. This work is the first example of a chemo-enzymatic cascade which combines in the same reaction medium MAO-N with a metal-catalyst, other than boron reducing agents. Finally, the aromatizing properties of MAO-D5 has been exploited for the synthesis of pyrrole 68 which has structural similarity to the novel class of antitubercular compounds described within chapter 2. Further investigation on the aromatizing property of MAOs biocatalysts toward other unsaturated heterocyclic rings (e.g. 3-pyrazoline, 1,4- dihydropyridine, 1,2-dihydropyridine) is highly recommended. Success in these biotransformations could be an important turning point for the development of more sustainable and competitive synthesis of building blocks for compounds of pharmaceutical interest.
5. Materials and Methods.